Nga Ombede et al. BMC Res Notes (2020) 13:358 https://doi.org/10.1186/s13104-020-05184-1 BMC Research Notes

RESEARCH NOTE Open Access Antimicrobial resistance and toxigenic profles of bacteria isolated from tropical shrimps ( notialis and monodon) in Cameroun Sabine Ninelle Nga Ombede1, Victorien Dougnon2* , Hornel Koudokpon2, Esther Deguenon2, Rajeunie Pernelle Jaelle Mindzie Ngomo2, Carine Tchibozo3, Jean Pierre Gnimatin2, François Tchoumbougnang1, Anges Yadouleton3 and Jacques Dougnon1

Abstract Objective: Post-harvest shrimp losses are a big problem due to the proliferation of spoilage bacteria. Presence and multiplication of these bacteria promotes the emergence of food-borne diseases. This study was carried out to characterize some spoilage bacteria from tropical brackish water shrimps and black tiger shrimps stored in ambient temperature (25 °C). Results: 22 isolates of Bacillus spp; 09 isolates of Coagulase Negative Staphylococci (CNS) and 04 isolates of entero- bacteria such as Pantoea spp (01); Serratia plymutica (01) and Serratia rubidaea (02) have been identifed. Resistance and virulence genes were then detected. All isolates expressed resistance to at least three of antibiotics tested. 03 isolates of enterobacteria were susceptible to cetfazidim and amoxicillin-clavulanic acid. Bacillus spp showed total susceptibility to cefxim, ertapenem and cetfazidim. Staphylococci were susceptible to clindamycin. Pantoea spp was resistant to all antibiotics but exhibited intermediate susceptibility to amoxicillin-clavulanic acid. 04 isolates of Staphy- lococci were positive to mecA resistances genes. All the enterobacteria harbor no tetracycline resistance genes. All the isolates of Bacillus exhibited the presence of enterotoxin genes. Also, a high prevalence of 21 isolates to hemolytic enterotoxins was noted. 17 isolates from them kept ability to cell-lyse factor production like sphingomyelinase activi- ties. The majority of Bacillus isolates identifed by the present study poses a potential risk of food poisoning due to the prevalence of toxin genes found. Keywords: Spoilage, Shrimps, Bacillus, Resistance genes, Enterotoxins

Introduction long-chain polyunsaturated fatty acids [1]. In Cameroun, Shrimps are great component of global seafood produc- tropical brackish water shrimp (Farfantepenaeus notialis) tion. In general, they contain good quantities of digest- and black tiger shrimp (Penaeus monodon) are ible proteins, essential aminoacids, bioactive peptides, widely consumed [2]. Shrimps are generally safe for con- sumption but their exposure to handling practices may occasionally entail health risks [3, 4]. Many retailers inap- *Correspondence: [email protected] propriately stored shrimps in addition to poorly handling 2 Research Unit in Applied Microbiology and Pharmacology of Natural practices causing postharvest degradation [5]. Specifc Substances, Polytechnic School of Abomey‑Calavi, University of Abomey- Calavi, Abomey‑Calavi, Benin spoilage organisms will change characteristics of food as Full list of author information is available at the end of the article consequence of contamination [6]. Many studies showed

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emergence of foodborne diseases due to spoilage bacteria Antibiotic disks (HIMEDIA, India) used were amoxicil- [7]. Gram-positive bacteria are slightly dominant in trop- lin (30 μg), amoxicillin-clavulanic acid (30 μg), cefxim ical warm water [8]. Tis suggests their great implications (5 μg), ceftazidim (30 μg), ceftriazone (30 μg), ciprofox- in spoilage, especially at ambient temperature [9]. Food- acin (5 μg), clindamycin (10 μg) colistin (25 μg), ertap- borne illness could result from the fact that those bacte- enem (10 μg), erythromycin (15 μg) fosfomycin (50 μg), ria harbor enterotoxins and resistance genes [7]. Te aim imipenem (10 μg), nalidixic acid (30 μg), oxacillin (1 μg), of this study was then to explore antibiotics resistance penicillin (10 μg), pristinamycin (15 μg) and vancomycin and virulence factor of spoilage bacteria isolated from (30 μg). shrimps stored in ambient temperature (25 °C) in our markets. Molecular identifcation of antibiotic resistance genes Main text DNA was extracted from the overnight colony obtained Materials and methods by spreading on Muller Hinton agar. DNA was extracted Collection and storage of samples from isolates using Qiagen blue extraction kit. PCR mix- ture of 23 µl contained 10 Nmole of genomic DNA, 0.5 µl Experiments were carried out with 5 kg of tropical brack- of each primer, 0.5 µl dNTP, 2.5 µl Bufer with MgCl2, ish water shrimp (Farfantepenaeus notialis) and the same 0.125 µl Taq DNA Polymerase, and 16 µl distilled water. quantity of black tiger shrimp (Penaeus monodon). Te For enterobacteria and staphylococci isolates, PCR detec- sampling took place from November to December 2018. tion was performed for the following diferent resistance Freshly harvested shrimp were purchased from ‘Youpwe genes : macrolide-resistance genes, vancomycin resist- fsheries market’ in Douala, Cameroon. Tose samples ance genes, methicillin resistance genes and tetracy- were transported to the laboratory in iced box. Tey cline resistance genes. PCR conditions included initial were then packed into four groups of 250 g portions per denaturation for 5 min at 94 °C, followed by 40 cycles of species in sterile plastic bags and kept at 20 °C until − denaturation for 45 s at 94 °C, annealing for 55 s at 45 °C, lab processing. Every 2 days, one plastic bag from each extension for 90 s at 72 °C and fnal extension for 10 min species was randomly taken for enumeration and isola- at 72 °C. PCR conditions for mecA were in accordance tion of total spoilage bacteria. Before this, shrimps were with those described by Igbinosa and Beshiru [11]. For removed from fridge and left at 25 °C for 24 h in the Bacillus, virulence and enterotoxins genes were investi- laboratory. Tis was done at room temperature because gated. Genes encoding hemolysin (hbl-D/A), non-hemo- shrimps are sold in the markets without cold conditions lytic enterotoxin (nheB), B. cereus enterotoxin T (bceT) and allowed spoilage odor to be produced. and enterotoxin FM (entFM) were screened. Additional Identifcation of spoilage bacteria fle 1: Table S1 show the primers used in the study. Virulence factors targeting genes coding for two phos- 25 grams of shrimp per specie were aseptically added to pholipases associated with cell lysis, sphingomyelinase 225 ml of peptone water and homogenized [10]. Seven (sph) and phosphatidylinositol-specifc phospholipase C fold dilutions of each homogenate were prepared and (piplc) were also investigated in accordance with Mata- 0.1 ml of 10­ −7 dilution was used for enumeration and iso- rante et al. [12] and Mohammadou et al. [13]. PCR prod- lation on nutrient agar. Replicate plates were incubated in ucts were analyzed on 1.5% (w/v) agarose gel stained (120 aerobiosis and in anaerobiosis at 37 °C. Tereafter count- volts at 35 min) with ethidium bromide (0.5 µg/ml) and ing, three characteristic isolates per plate were purifed visualized by UV. Additional fle 1: Table S2 shows the on Mueller Hinton. Pure isolates were stored at 80 °C − primers of virulence factor genes detection. in 20% glycerol. Identifcation of bacterial isolates was then carried out using biochemical tests. Results Detection of resistance profle Identifcation of spoilage bacteria An overnight bacterial pre-culture was diluted to obtain Shrimps contained more than 10­ 7 germs/g after 24 h in a turbidity of 0.5 McFarland. Kirby Bauer techniques ambient storage. Additional fle 1: Figure S1 shows the were used to perform susceptibility testing (Antibiogram microbial population present in tropical shrimps. Results Committee of the French Society of Microbiology, CA- showed the main presence of Gram-positive bacteria SFM, 2012). Broth cultures were aseptically swabbed on such as Bacillus spp and Coagulase Negative Staphylo- Mueller Hinton agar. Antibiotic disks were placed on the cocci (CNS). Some isolates of enterobacteria (Serratia agar plates and incubated 24 h at 37 °C. Inhibition zone spp and Pantoea spp) were also identifed. diameters were measured after 24 h. Susceptibility or resistance was determined according to CA-SFM (2012). Nga Ombede et al. BMC Res Notes (2020) 13:358 Page 3 of 6

Resistance profle Resistance and enterotoxins‑virulence genes All the isolates of Coagulase Negative Staphylococci Bacillus isolates isolated harbor at least two enterotoxins were susceptible to clindamycin. Tey showed total genes. Only one isolate was positive to hemolysin gene. resistance to oxacillin and penicillin. Te isolates were Tree isolates were negative to sph and piplo virulence resistant to ciprofoxacin, colistin and vancomycin genes. All Bacillus isolates (08) harbor Bacillus cereus (55.56%), to nalidixic acid (88.89%). 15 isolates showed enterotoxin T and also the sph gene. 04 isolates of Staph- resistance to ceftazidim, ceftriaxon, cefxim and amoxi- ylococci exhibited the presence of MecA resistant gene. cillin. Table 1 presents the antimicrobial susceptibility All enterobacteria isolates were tested negative to tetra- profle of Staphylococci. cycline resistance gene. Figure 1 shows the results of PCR Enterobacteria showed total resistance to ceftriazon, product migration. cefxim, amoxicillin and ertapenem. Only isolates of Pantoea spp showed intermediate resistance to amoxi- Discussion cillin—clavulanic acid and total resistance to all other Identifcation of spoilage bacteria antibiotics tested. Bacillus spp were susceptible to imi- Tis study was carried out to explore resistance and vir- penem. 59.09% of Bacillus spp exhibited intermediate ulence genes of spoilage bacteria isolated from shrimps resistance to ciprofoxacin and amoxicillin-clavulanic in Cameroun. Results of this study are similar to those acid. Table 2 shows antimicrobial susceptibility profle of Dabadé et al. [9] who mainly found positive Gram of enterobacteria and Bacillus spp. bacteria, especially lactic bacteria and enterobacteria in black tropical brackish water shrimp. Tose Authors revealed that potentially spoilage bacteria in tropical shrimps in Benin may be H­ 2S-producing dominant group of bacteria. Finding in this study suggests that Bacillus Table 1 Antimicrobial susceptibility of Staphylococci and Staphylococci species are responsible for spoilage detected in tropical shrimp. Te results of this study are Antibiotics S if R if < S % I % R % ≥ diferent from those of Srinivasan and Saranraj [14] who Ciprofoxacin 25 22 3 (33.33%) 1 (11.11%) 5 (5.56%) found Vibrio cholerae, Pseudomonas fuorescens, Salmo- Pristinamycin 22 19 5 (55.56%) 1 (11.11%) 3 (33.33%) nella Typhi, Staphylococcus aureus and Escherichia coli. Penicillin 29 18 0 0 9 (100%) Tese microorganisms are common in polluted water, Fosfomycin 14 14 6 (66.67%) 0 3 (33.33%) so it is possible to fnd them in the seafood. Te results Erythromycin 22 17 5 (55.56%) 1 (11.11%) 3 (33.33%) obtained are comparable to those of Al Bulushi et al. [8] Clindamycin 15 15 9 (100%) 0 0 who who also isolated Staphylococci in their study. Tey Nalidixic Acid 20 15 1 (11.11%) 0 8 (88.89%) also related that the frequency of Staphylococci in marine Vancomycin 17 – 4 (44.44%) 0 5 (55.56%) environments depend on geographic location. Bacillus Oxacillin 21 21 0 0 9 (100%) species in coastal fsheries are also common, despite the Colistin 15 15 4 (44.44%) 0 5 (55.56%) relative abundance of Staphylococcus spp [8]. Abundance of Bacillus in coastal waters and marine fsheries depends S sensible, R resistant, I intermediate

Table 2 Antimicrobial susceptibility of enterobacteria and Bacillus species Antibiotics S if R if < Enterobacteria Bacillus spp ≥ S I R S % I % R %

Amoxicillin - clavu- 23 16 3 1 – 8 (36.36%) 13 (59.09%) 1 (4.55%) lanic Acid Vancomycin 17 – 2 – 2 7 (31.82%) – 15 (68.18%) Ceftazidim 21 19 – 1 3 – – 22 (100%) Impenem 21 18 3 – 1 22 (100%) – – Ciprofoxacin 25 22 1 2 1 6 (27.27%) 6 (27.27%) 10 (45.45%) Ceftriazone 26 23 4 0 1 (4.55%) 21 (95.45%) Cefxim 25 22 4 0 0 22 (100%) Amoxicillin 23 16 4 0 1 (4.55%) 21 (95.45%) Ertapenem 28 26 4 0 0 22 (100%)

S sensible, R resistant, I intermediate Nga Ombede et al. BMC Res Notes (2020) 13:358 Page 4 of 6

Fig. 1 PCR gene migration

on temperature, depth and decline distance from the Resistance and enterotoxins‑virulence genes coast and, suggests that Bacillus species in coastal water Tetracycline resistance genes can be explored to current originate from runof [15]. Mohammadou et al. [13] and emerging multidrug-resistant pathogens including reported Bacillus species such as Bacillus cereus, Bacil- carbapenem-resistant Enterobacteriaceae. None entero- lus thuringiensis and Bacillus anthracis were well-known bacteria presented tetracycline resistance genes, although as food poisoning in spoilage food. Gram-positive bacte- they were all resistant to ertapenem. MecA genes as pre- ria were more abundant than Gram-negative in tropical sented in this study about Staphylococci are similar to water. According to Al bulushi et al. [8], their infuence those of Ali [23] and Igbinosa and Beshiru [11]. MecA in spoilage remain little probed. Findings of this study gene is present in all Staphylococci isolates and is known also showed low frequency of enterobacteria such as Ser- to encode penicillin binding protein 2a (PBP2a). Beta-lac- ratia and Pantoea isolates who grow in a broad range of tam resistance is mostly attributed to mutations in mecA temperatures and substrates according to Garrity et al. gene, but other genetic elements may also be considered [16]. Many species related with food spoilage, have been for the explanation of the mechanism of resistance. Half described as opportunistic human pathogens [17–19]. of Bacillus isolates were positive to HBL genes. Tese results are similar to those of Chaves et al. [7]. Te same Antimicrobial susceptibility profle authors demonstrated prevalence of hemolytic toxins in Bacillus isolates from fermented foods. Many authors cor- Te presence of Pantoea spp and Serratia spp in food is relate prevalence of enterotoxins HBL-NHE [24]. Accord- often neglected, when compared to classic multidrug- ing to Trans et al. [25], wall peptidase (sph genes and resistant Gram-negative pathogens [20–22] likewise in PIPLC genes) are involved in adhesion, bioflm formation this study. Tey are opportunistic pathogens among the and virulence. Prevalence of entFM genes in more than most common causes of nosocomial diseases and trans- half of Bacillus isolates is located on the chromosome missible by food ingestion. Resistance diversity of Bacil- and appears to be common to Bacillus thuringiensis and lus isolates in the study is not similar to the works of B. cereus. Detection of EntFM is similar to the results of Mohammadou et al. [13]. Tose authors revealed rela- Mohammadou et al. [13]. Presence of Bcet gene is evi- tive susceptibility of Bacillus isolates to antibiotics unlike dence of presence of B. cereus among isolates. Agata et al. our results. Chaves et al. [7] also found susceptibilities of [26] also demonstrated that prevalence of this gene carried Bacillus isolated from Brazilian food to gentamicin and out other isolates than B. cereus. Te majority of the iso- tetracycline. Drug resistance of isolates in this study may lates isolated pose a potential risk of food poisoning due be attributed to the spores of Bacillus species. Moham- to their antibiotic resistance profle and the prevalence of madou et al. [13] found that B. subtilus was able to pro- toxin genes found in Bacillus isolates. Te surveillance of duce bacteriocins such as subtilin in Mbuja. Nga Ombede et al. BMC Res Notes (2020) 13:358 Page 5 of 6

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